Major histocompatibility complex (MHC) class I molecules include highly polymorphic classical HLA class Ia (HLA-A: 1729 alleles with 1,264 proteins; HLA-B: 2329 alleles, 1786 proteins; and HLA-Cw: 1291 alleles, 938 proteins) and least polymorphic non-classical HLA-Ib (HLA-E: 10 alleles, 3 proteins; HLA-F: 22 alleles, 4 proteins; and HLA-G: 47 alleles, 15 proteins), based on information published in October 2011 in EBML-EBI website at www<dot>ebi<dot>ac<dot>uk</>imgt</>hla</>stats<dot>html.
Each HLA molecule consists of a heavy chain (HC) of about 346 amino acids in length. An HC consists of three extracellular domains (α1, α2 & α3), a transmembrane domain and a C-terminal cytoplasmic domain. In some cases, the HC is non-covalently linked to β2-microglobulin (“β2 m”), which is about 99 amino acids in length.
HLA-E was first discovered in 1987; see, for example, Geraghty et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 9145-9149; and Koller et al., 1988, J. Immunol. 141: 897-904. One of the functions of HLA-E is to present peptides to CD8+ T-lymphocytes. The peptides presented include but are not limited to (1) All leader sequence peptides of HLA-Ia antigens, namely HLA-A, HLA-B and HLA-Cw and (2) peptides from (a) Heat Shock Proteins (Hsp-60), (b) cytomegalovirus (CMV), (c) Epstein Barr Virus (EBV); (d) Influenza virus, (e) Salmonella enteric and (f) Mycobaterium glycoproteins (Iwaszko and Bogunia-Kubik, 2011 Arch Immunol Ther Exp. 59(5):353-367).
The HLA-E gene is expressed in resting T-lymphocytes. It is also commonly expressed by cells such as endothelial cells, immune cells (B-, T-lymphocytes, NK cells, monocytes and macrophages), and trophoblasts. Most importantly, HLA-E is overexpressed in tumor cells, possibly caused by malignant transformation of human tissues. See, for example, Marin R et al., 2003 Immunogenetics. 54:767-775; Wischhusen J et al., 2005, J Neuropathol Exp Neurol. 64:523-528; Derré L et al., 2006. J. Immunol. 177:3100-3107; Mittelbronn, et al., 2007 J. Neuroimmunol. 189: 50-58; Goncalves et al. 2008, Stangl et al., 2008, Cell Stress Chaperones 13(2):221-230; Levy et al., 2009, Innate Immun. 15(2):91-100; Hanak L et al., 2009, Med Sci Monit. 15(12):CR638-643; Sensi M, et al., 2009, Int Immunol. 21(3):257-268; de Kruijf E M et al., 2010 J. Immunol. 185:7452-7459; Kren L et al., 2010 J. Neuroimmunol. 220:131-135; Kren L et al., 2011 Neuropathology 31:129-134; Kren L et al., 2012 Diagnostic Pathology 7:58; Kren L, et al., 2012 Pathology: Research and Practice 208: 45-49; Allard M et al., 2011 PLoS One. 6(6):e21118; Benevolo M, et al., 2011, J Transl Med. 9:184; Gooden M et al., 2011, Proc Natl Acad Sci USA. 108:10656-10661; and Silva T G et al., 2011, Histol Histopathol. 26:1487-1497; each of which is hereby incorporated by reference herein in its entirety.
Increased cellular expression of HLA-E induces the release of HLA-E in circulation (e.g., Derré L et al., 2006. J. Immunol. 177:3100-3107). For example, soluble HLA-E (sHLA-E) is found in the sera or plasma of patients with immune-mediated vascular diseases, Kawasaki Disease, a systemic pediatric vasculitis, as well as in normal individuals (e.g., Lin et al., 2009 Arthritis & Rheumatism 60(2): 604-610).
The soluble HLA-E (sHLA-E) may be found without β2m. In intact HLA-E, the presence of β2m can mask some of the peptides sequences of the sHLA-E heavy chain that would be otherwise exposed and become immunogenic. In other words, some of the peptide sequences of sHLA-E have lost immunogenic capacity due to association with β2m.
Several monoclonal antibodies to HLA-E are available commercially. They include MEM-E/02, MEM-E/06, MEM-E/07, MEM-E/08, mAb 3D12 and mAb DT9. These anti-HLA-E monoclonal antibodies were used for cancer diagnosis based on their assumed specificity for HLA-E. See, for example, Shimizu et al., 1988, Proc Natl Acad Sci USA. 85:227-231; Menier et al., 2003, Hum Immunol. 64(3):315-326; Gooden M et al., 2011, Proc Natl Acad Sci USA. 108:10656-10661; Stangl et al., 2008, Cell Stress Chaperones 13(2):221-230; Allard M et al., 2011 PLoS One. 6(6):e21118; Levy et al., 2009, Innate Immun. 15(2):91-100; and Sensi M, et al., 2009, Int Immunol. 21(3):257-268; each of which is hereby incorporated by reference herein in its entirety.
These known anti-HLA-E monoclonal antibodies, however, have been shown to cross-react with other antigens. For example, Ravindranath et al. showed that MEM-E/02 antibodies bind A*2402, B*1301, B*1401, B*1502, B*1513, B*1801, B*3501, B*3701, B*4001, B*4006, B*4101, B*4403, B*4501, B*4601, B*5601, B*7301, B*7801, B*8201, Cw*0102, Cw*0304, Cw*0501, Cw*0602, Cw*0701, Cw*1802 at 1/300 dilution. In the same study, MEM-E/06 antibodies were shown to bind B*1401, B*4006, B*4101, B*8201, Cw*0501, Cw*0802, Cw*0701, Cw*1802 at 1/300 dilution. MEM-E/07 and E/08 also antibodies were to bind B*1301, B*3801, B*4006, B*4101 (E/07 only), B*8201 (E/07 only), Cw*0501, Cw*0701, Cw*1802 at 1/300 dilution. MEM-E/07 and MEM-E/08 were shown to react reasonably well with HLA-G. In addition, an anti-HLA-E murine mAb 3D12 also reacted with several HLA Class Ia alleles. See, for example, Ravindranath et al., 2010, Mol. Immunol. 47: 1121-1131 and Ravindranath et al., 2010, Mol. Immunol. 47.1663-1664. Further, it was reported that yet another anti-HLA-E mAb, mAb DT9 strongly reacted with HLA-A*8001, N*1301, B*3501, B*4006 and B*7301. See, e.g., Shimizu et al., 1988, Proc Natl Acad Sci USA. 85:227-231; which is hereby incorporated by reference herein in its entirety.
The cross reactivity of anti-HLA-E mAbs to HLA-A, -B or -Cw is possibly due to recognition of shared epitopes found between HLA-E and HLA-Ia alleles (Table 1). See, Ravindranath et al., 2010, Mol. Immunol. 47. 1663-1664; and Ravindranath et al., 2010, J. Immunol. 185: 1935-1948.
CD94 and NKG2a receptors are present on CD8+ T lymphocytes and Natural Killer T Cells. When an HLA-E binds to CD94 and NKG2a receptors on CD8+ T cells and NKT cells, incoming activation signals of T cells are dampened by recruitment of phosphatases like SHP-1 to the signal transducing synapse, which results in decreased effector functions (e.g., Rodgers and Cook, 2005, Nat Rev Immunol 5:459; Chang W C et al., 2005, Int J Gynecol Cancer 15:1073; and Lanier L L, 2005, Annu Rev Immunol 23:225). In other words, in the absence of activating signals, the CD8+ cells remain paralyzed, unless the proliferation of activated CD8+ T cells are augmented to exceed interaction with HLA-E expressing cells.
In contrast to overexpression of HLA-E, loss of MHC class Ia expression is known to occur in several cancers including primary and metastatic melanoma. Thus, loss of MHC class Ia expression and increased CD94/NKG2-A/B expression are linked with tumor progression (Vetter et al., 2000 J. Invest. Dermatol. 114: 941-947).
In general, the events taking place in the tumor microenvironment can be summarized as follows: CD8+ Cytotoxic T cells (CTLs) and NKT cells infiltrate tumor tissue to destroy tumor cells. CD8+ CTLs release IFN-γ after infiltrating into tumor cells. IFN-γ induces overexpression of HLA-E. HLA-E epitopes functions as a major ligand for CD8+ Cytotoxic Lymphocytes (CTL) and the Natural Killer T cell (NKT) inhibitory receptor CD94/NKG2A (FIGS. 1A and 1B). CD8+ T cells with CD94/NKG2A are more in tumor tissues than in peripheral blood. Both overexpression of CD94, NKG2a & HLA-E may vary with the stages of cancer, from primary to lymph node & organ metastasis.
Survival curves in ovarian cancer have been compared in relation to the expression of HLA-E in tumor and CD8+ on T cells (Gooden M et al 2011. Proc. Natl. Acad. Sci. U.S.A. 108(26):10656-10661). Patients with high levels of tumor infiltrating CD8+ T cells survive significantly (p<0.04) higher than those with low CD8+ T cells. The survival of patients is significantly (p<0.001) higher when high CD8+ T cells co-exist with tumor cells with low level of expression of HLA-E. However, the survival of patients with high CD8+ T cells are much lowered to the level of low CD8 T cells.
One of the salient strategies to overcome HLA-E-mediated inactivation of CTLs/NKTs is to block HLA-E on the tumor cell surface with antibodies designed to block only and specifically HLA-E. These antibodies have the potential to prevent with the ligand-receptor interaction between tumor cells expressing HLA-E and CD94 and NKG2a receptors on CD8+ T cells and NKT cells.
Whether HLA-E blocking HLA-E specific monoclonal antibodies are capable of any other immunomodulatory functions deserves to be elucidated before chimeric or humanized monoclonal antibodies are introduced into the patients.
What is needed are truly monospecific antibodies that that have no reactivity to other human leukocyte antigen (HLA) class I antigens except HLA-E. Such monospecific antibodies would be more reliable and invaluable for immunodiagnosis of HLA-E in normal and pathological tissue samples overexpressing HLA-E.